作者：HongWei Liu
作者单位：Department ong>ofong> Ophthalmology, Beijing Tongren Hospital Affiliated to Capital Medical University, Beijing 100730, China Department ong>ofong> Pharmacology, Beijing Institute ong>ofong> Ophthalmology, Beijing 100005, China
【摘要】AIM: To study the expression ong>ofong> ong>basicong> ong>fibroblastong> ong>growthong> ong>factorong> (bFGF) ong>receptorong> ong>proteinong> in human lens epithelial cells (HLECs).
METHODS: Immunohistochemistry was used to detect the level ong>ofong> bFGF ong>receptorong> ong>proteinong> and image analysis was adopted to perform the relative quantitative analysis on it.
RESULTS: There was bFGF ong>receptorong> ong>proteinong> in HLECs accordingl to both qualitative and quantitative analysis.
CONCLUSION：bFGF ong>receptorong> ong>proteinong> exists in HLECs and it is the material foundation for bFGF to improve the proliferation ong>ofong> HLECs.
KEYWORDS: human lens epithelial cells (HLECs); ong>proteinong>; ong>basicong> ong>fibroblastong> ong>growthong> ong>factorong> ong>receptorong>; immunohistochemistry
【关键词】 human lens epithelial cells (HLECs) ong>proteinong>; ong>basicong> ong>fibroblastong> ong>growthong> ong>factorong> ong>receptorong> immunohistochemistry
INTRODUCTION
With the continuous development ong>ofong> molecular biological techniques, the effects ong>ofong> ong>basicong> ong>fibroblastong> ong>growthong> ong>factorong> (bFGF) and its ong>receptorong> on human lens epithelial cells (HLECs) became increasingly known by researchers. It is hopeful to control the amount ong>ofong> its ong>proteinong> and the biological effects by using its antagonist or synergist in the future. Our previous study [1] has confirmed that there is the expression ong>ofong> bFGF ong>receptorong> gene in HLECs. The aim ong>ofong> this study is to clarify whether bFGF ong>receptorong> ong>proteinong> exists in HLECs and its quantity in HLECs.
MATERIALS AND METHODS
Preparation ong>ofong> Specimen Paraffin section: fresh human eyeballs were fixed in 40g/L buffered formalin after the sclera was cut open. They were cut into 3 4μm sections after dehydration and paraffin embedding.
Cell smears Cell suspension was coated onto glass slides and then the slides were kept at 20℃.
Lens capsule slides The anterior lens capsules removed in operation were coated onto slides with cell side up. The slides were dried in air at room temperature and then put in 700mL/L ethanol.
Immunohistochemistry (ABC method)
Main reagents Mouse IgG monoclonal antibody (human bFGF specific ong>receptorong>) (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and ABC Kit (Vector Laboratories, Peterborough, UK).
Staining procedures Paraffin sections were dewaxed to distilled water, and cell smears and capsule slides were put in distilled water directly. They were rinsed with PBS for 5 minutes and with 3mL/L hydrogen peroxidemethanol for 30 minutes to eliminate endogenous peroxidase, then with distilled water for 3 minutes twice and with PBS once. The slides were put in a container filled with citrate buffer, and the container was put in a microwave oven to be heated for 10 minutes. The container was took out and cooled at room temperature for 10 minutes. The slides were washed with PBS for 5 minutes three times and then were transfered to a wet box. The slides were sealed with calf serum (1∶50) and incubated for 10 minutes at room temperature. Redundant serum was removed and diluted specific antibody was added on the slides at 4℃ overnight. The slides were washed with PBS for 5 minutes three times, incubated in biotinlabeled second antibody (diluted with PBS) for 30 minutes at 37℃ and were washed again with PBS for 5 minutes three times. The slides were incubated with the mixture ong>ofong> A and B (1∶100) at 37℃ for 45 minutes, washed with PBS for 5 minutes three times, colordeveloped with DABH2O and observed under microscope. The reaction time was controlled. After the color development, the slides were restained with hematoxylin, and the slides were observed under light microscope after dehydration, transparency and mounting.
Result evaluation ong>ofong> immunohistochemistry The positive cells were brownish yellow or brownish black in ABC method. (Negative control: PBS was used as blank control or normal rabbit serum as substitution control).
Image Analysis Experimental apparatus: Cambridge Quantimet 970 image analyzer (Cambridge Instruments. Ltd, Cambridge, UK) was used.
Image analysis A total ong>ofong> 2050 cells were selected from each ong>ofong> the positive and negative slides in immunohistochemistry as statistical targets. Microscope and pickup head were used in the acquisition ong>ofong> images. The magnification ong>ofong> the microscope: field lens 40×, projective amplification 2.0×, circuit amplification 20× and the total amplification 1600×. The scale value for one pixel was 0.1695μm. The microscope ruler and shadow were verified, the slides were loaded onto objective table and the focusing was made to show the image as clearly as possible.
Table 1 Quantitative results ong>ofong> fetal(5 months)human lens epithelial cells(HLECs)(paraffin) Immunohistochemical morphometry（略）
△P>0.05,bP<0.01
Table 2Quantitative results ong>ofong> adult human lens epithelial cells (HLECs)(capsule) Immunohis tochemical morphometry（略）
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